Medicinal products for the treatment of blood coagulation disorders

ABSTRACT

A virally safe, thrombin-free factor-XIa concentrate or a coagulation factor concentrate which contains factor XIa as an active pharmaceutical ingredient and which is obtained by fractionation of plasma or serum or by genetic engineering and is suitable for the treatment of coagulation disorders attributable to diminished and/or delayed thrombin formation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/254,451, which is the national stage of International Application No.PCT/AT2010/000339, filed Sep. 16, 2010, which claims priority toEuropean Application No. 09450173.1, filed on Sep. 16, 2009. Theforegoing applications are incorporated herein by reference.

BACKGROUND OF INVENTION

The present invention relates to a virally safe, thrombin- andpyrogen-free factor-XIa concentrate and to the use of a virally safefactor-XIa concentrate for the production of a pharmaceuticalcomposition for the treatment of coagulation disorders.

Injuries of the organism may result in severe bleeding, with massive oreven fatal blood loss. The organism is protected against severe bloodloss by a hemostatic mechanism, which causes the exiting blood tocoagulate, whereby the bleeding is controlled and the wound is closed.Research into the hemostatic process has been conducted during the lastcentury, and although a basic understanding has been gained, is stillongoing.

According to the present understanding, disruption of vessels exposessubstances which trigger blood coagulation, so that a hemostatic plugcan be formed. The plug contains a network of fibrin and platelets, theplatelet count exceeding the blood count 20 to 200-fold. The plugadheres to, and closes, the injured site. The importance of the role ofplatelets in hemostasis can be illustrated by the fact that patientswith afibrinogenemia do not tend to bleed profusely, while patients withsevere thrombocytopenia may suffer non-controllable bleedings.

The blood coagulation process involves individual blood components suchas blood cells, microparticles, and blood plasma. The coagulation ofblood occurs when fibrinogen as a soluble protein in the blood plasma isconverted into insoluble fibrin by the enzymatic action of thrombin, anenzyme which splits fibrinogen into soluble fibrin monomers andfibrinopeptides A and B. The fibrin monomers aggregate to fibrin monomercomplexes and finally to insoluble fibrin. In patients with normal bloodcoagulation, only approximately one fourth of the fibrinogen that waspresent in the amount of blood from which a clot is formed, is convertedinto fibrin. The further conversion of fibrinogen in the clot depends onthe amount of thrombin generated therein (Kumar R, et al.)

Thrombin results from prothrombin by activation at the end of the enzymecascade, where coagulation factors which are pro-enzymes are activatedinto activated coagulation factors which are enzymes in a predeterminedorder. Coagulation factors which are not pro-enzymes are pro-co-factors,which are converted enzymatically into co-factors. Each co-factorenhances the enzymatic conversion of a specific pro-enzyme into anenzyme (Mann KG, et al.).

The enzyme cascade which results in thrombin can be divided into fourdifferent pathways, the extrinsic, the intrinsic, and the commonpathway, and in addition, the so-called contact-phase. The presentunderstanding is that on an injured blood vessel, tissue factor, acell-bound lipoprotein, initiates the extrinsic pathway by forming acomplex with factor VIIa, and this complex activates factor X intofactor Xa. Factor Xa forms another enzyme complex, calledprothrombinase, which generates thrombin from prothrombin. This pathway,where factor Xa generates thrombin, is called the common pathway. Thegeneration of factor Xa by the extrinsic pathway is soon interrupted bytissue factor pathway inhibitor. As a consequence, only small amounts ofthrombin can be generated via the extrinsic and common pathways. Thesmall amounts of thrombin, however, trigger the activation of theintrinsic pathway. By activation of the intrinsic pathway large amountsof factor X are activated, so that thrombin is generated in excess viathe common pathway. To what extent platelets and the contact phasecontribute to the activation of the intrinsic pathway, is still a matterof discussion (Walsh P).

In patients who suffer from severe coagulation disorders all theirlives, even minor traumatic events may cause uncontrollable bleedings.Such patients have either inherited deficiencies of specific coagulationfactors or acquired the deficiencies in the course of their lives.Bleeding disorders of this type are referred to as hemophilia. Mostpatients who suffer from severe hemophilia have a deficiency ofcoagulation factor VIII (Brown S).

Coagulation factor VIII is a pro-cofactor, which is enzymaticallytransformed into a cofactor via the intrinsic pathway. This cofactoraccelerates the activation of factor X by tenase, an activatedfactor-VIII-IX complex, dramatically, so that an excess of thrombin canbe formed via the common pathway. Thrombin converts fibrinogen andfactor XIII into fibrin and factor XIIIa, respectively. Factor XIIIa, atransglutaminase, causes the formed fibrin to cross-link, which resultsin an increased adhesion of the coagulated blood to the wound edges. Inthe further course, TAFI, a proenzyme, is activated into TAFIa. TAFIasplits off the receptor peptide for plasmin from fibrin, rendering thecross-linked fibrin more resistant against lysis. These enzymaticprocesses lead to an increased stability of the clot, an increase in itselasticity, and an increase in its resistance to lysis.

Patients with factor VIII deficiency form hemostatic plugs withdifficulty only. If they do form a plug, the plug is fragile and willdissolve within short by fibrinolytic processes, the reason being thatthe intrinsic pathway is impaired in the clot and only an insufficientamount of thrombin is generated in the clot (Sixma J, et al.).

Once it became possible to produce concentrates of coagulation factorVIII as part of the fractionation of human plasma, bleeders with factorVIII deficiency could be treated so successfully that their averagelifespan of 15 years could be prolonged to that of a normal person.

It was gradually understood that a considerable percentage of patientsthat had been treated successfully with factor VIII concentrates turnedrefractory to the very products they were treated with. Investigationsof the phenomenon led to the conclusion that those patients developedantibodies against the homologous factor VIII they had received, whichlargely inhibited the function of factor VIII in the coagulationprocess. Such bleeders, thereafter referred to as factor VIII inhibitorpatients, suffered the same fate as factor VIII deficient patients priorto the availability of factor VIII concentrates (van den Berg H, etal.). The same pathological process occurs in factor IX deficientpatients as they become refractory to substitution therapy with factorIX concentrates.

In the 1970ies, factor VIII inhibitor patients were treatedunsuccessfully with different hemostatic medicinal products. It becameknown that only certain batches of products containing prothrombincomplex were effective. Medicinal products containing prothrombincomplex contain several vitamin-K-dependent coagulation factors,predominantly factors II, IX, X, and VII, and, depending upon themanufacturing process, those factors may in part be present in activatedform (Kelly P, et al. and Kurczynski E, et al.). The Baxter Group thensucceeded in manufacturing such partly activated prothrombin complexpreparations and in marketing them by the trade name of “Autoplex”(Fekete L, et al.).

About the same time, Immuno AG succeeded in developing a product for thetreatment of the same group of patients, which bridges the impairment inthe coagulation cascade and normalizes coagulation (Eibl J, et al. andTurecek P, et al.). This product was introduced by the name of “FEIBA”.After the Baxter Group acquired Immuno AG in 1998, Baxter discontinuedthe production of Autoplex and continued producing and distributingFEIBA worldwide.

In the early 1980's, Novo Nordisk NS introduced a recombinant factorVIIa product (Hedner U, et al.) by the name of “NovoSeven” (Hedner U)for the treatment of patients with factor VIII and factor IX inhibitorsand other bleeding disorders. (Roberts H, et al.) Thus, two companiesbecame leaders in parenteral medicinal products for the treatment ofbleeders with inhibitors. As far as estimates allow, about 90% of themarket today is covered by FEIBA and NovoSeven, amounting to annualsales of 1.5 billion Dollars, with NovoSeven accounting forapproximately 60% and FEIBA approximately 40%.

The most recent publication about the efficacy of FEIBA and NovoSeven asassessed in a comparative multicenter trial reports no significantdifference in the percentage of patients who stopped bleeding within sixhours of treatment. Neither has their been a difference in the rate ofadverse events, particularly thromboses (Astermark J, et al.).

Jan Astermark et al have shown that treatment with those productsproduced rapid hemostasis in only half of the patients. In about 25percent of the patients, hemostasis occurred only after repeated dosesof either Feiba or NovoSeven, and only after a prolonged period of time.Another 25 percent had either insufficient hemostasis, or the bleedingcould not be controlled at all. The frequency of adverse events,particularly thromboses, was the same in either group.

Since the introduction and successful use of these pharmaceuticalpreparations, there has been speculation about their mode of action. Inparallel, and irrespective thereof, the understanding of the coagulationprocess itself has changed and improved over the last 30 years.Nevertheless, no uniform or ultimate opinion has been reached abouttheir mode of action (Roberts H, et al.).

SUMMARY OF INVENTION

The objective of the invention is to provide a medicinal product forparenteral use which is efficacious in achieving fast and sustainedcontrol of bleeding in patients with coagulation disorders, and inparticular, patients with inhibitors to factors VIII or IX, and one thatwill be safer than the products presently on the market in terms ofadverse events, even if given in high doses. In addition, the cost ofproducing it should not be higher than those of the products presentlyon the market. The improvement of the coagulation process in thesepatients should consist not only in a shortening of the coagulation andbleeding times but be primarily achieved in the clot itself, where anamount of thrombin must be generated to compare with that in a healthyperson.

For a product to be administered parenterally, it is important that itcontains no thrombin activity and/or does not generate thrombin prior toadministration. Pure factor XI concentrates do not contain thrombin anddo not generate thrombin either during storage nor during freeze-drying,reconstitution or prior to administration. When other, non-activated andactivated coagulation factors are added for formulation, great careneeds to be used to maintain this absence of thrombin. The latter is ofutmost importance for the safety of the product, the parenteralapplication of thrombin bearing the potential of creating thromboses orDIC. When thrombin is manufactured for topical application, factor XI orfactor XIa may be be used in the process of manufacture and may, if sodesired, be removed in the further course of manufacturing without thetopical activity of thrombin affecting hemostasis in any negativemanner.

In most patients suffering from hemophilia, be it because of a lack offactor VIII or factor IX, the intrinsic coagulation pathway is stronglydown-regulated. This is particularly true of patients who haveantibodies against one or the other of these factors. In persons withnormal blood coagulation, more than 90% of the thrombin is generated bythe intrinsic pathway, three quarters of the thrombin being formed inthe clot itself. In patients with hemophilia, the amount of thrombingenerated in the clot is insufficient, so that they cannot form bloodclots which persist for an extended period of time as would be necessaryto sustain hemostasis.

According to the invention, an alternative intrinsic pathway can begenerated by activated factors of the contact phase in the presence offibrin and/or fibrin monomer complexes. When factor XI, completely orpartially activated, is added to blood, platelet-rich or platelet-poorplasma of patients with hemophilia, thrombin generation in the clot isnormalized. This improves the quality of the formed clot considerably,which reflects in an increase in the elasticity module and theresistance against lysis.

Virus inactivation by solvent/detergent is best accomplished prior tochromatographic purification of the factor or factors, so that the addedsolvent/detergent can be separated. Further virus removal can beachieved by nanofiltration, ultrafiltration, and/or heating infreeze-dried state. The virus safe activated, partially activated ornon-activated factors or co-factors and their derivatives are thenstored at refrigerator temperature or deep-frozen prior to formulationof the medicinal product.

To stabilize factors XII and XI and their derivatives, high molecularweight kininogen, a co-factor of the contact phase, preferablybradykinin-poor kininogen, is added. In the same manner, the long chainhigh molecular weight kininogen split off by reduction, can be used.

In order to accelerate the coagulation process and hemostasis, activatedand non-activated factors of the prothrombin complex may be added duringformulation.

Such activated or non activated clotting factors as well as formulatedFactor XIa concentrates must be free of thrombin or must not generatethrombin prior to application.

Treatment with factors, in particular with activated factors of thecontact phase, may be accompanied by a risk of thrombosis. To reducethis potential risk, low avid serpins or other appropriate homologousprotease inhibitors are added to the coagulation factor concentrates ofthe contact phase to prevent activation during storage and/orapplication.

The addition of low-avid serpins or other appropriate proteaseinhibitors may prevent autoactivation of non-activated coagulationfactors, to the extent present in the medicinal product, during storageand/or application.

Therefore the invention is directed to a thrombin-free factor-XIaconcentrate or a thrombin-free coagulation factor concentrate containingfactor XIa for the manufacture of a pharmaceutical preparation whichdoes not form thrombin during storage, and for the manufacture of aparenterally applicable drug product which does not form thrombin duringor after freeze-drying, storage and reconstitution.

A preferred embodiment of the pharmaceutical preparation according tothe present invention contains serpins and/or high molecular weightkininogen or its split products.

A further proeferred embodiment of the pharmaceutical preparation isnanofiltered to improve viral safety, either before or afterformulation, preferably by nanofilters with a pore diameter of ≦20 nm oris filtered by a 1000 kDalton ultrafilter.

The pharmaceutical preparation according to the invention is preferablyrendered virally safe by treatment with solvent/detergent and subsequentremoval of solvent and detergent.

The pharmaceutical preparation according to the invention may be filledunder sterile and pyrogen-free conditions and may be freeze-dried andvirally inactivated by heating the filled, freeze-dried pharmaceuticalpreparations at no less than 90° C. and no more than 140° C. for 30 to180 minutes.

The invention is also directed to a thrombin-free factor-XIa concentrateor thrombin-free coagulation factor concentrate containing factor XIa,characterized in that it is obtained by fractionation of plasma or serumor by genetic engineering.

The thrombin-free factor-XIa concentrate or thrombin-free coagulationfactor concentrate containing factor XIa according to the invention orthe pharmaceutical preparation according to the invention are preferablyvirally safe and pyrogen-free.

The invention is also directed to a diagnostic kit for the determinationof the amount of factor-XIa concentrate which is necessary to normalizeprolonged blood coagulation in a reference factor-VIII inhibitor plasmaand in blood samples of patients with coagulation disorders.

The thrombin-free factor-XIa concentrate or the thrombin-freecoagulation factor concentrate containing factor XIa according to theinvention as described above can be used as an active ingredient for theproduction of a medicinal drug product for the treatment of coagulationdisorders.

BRIEF DESCRIPTION OF FIGURES

Thrombelastographic determination of coagulation defects and theirnormalization is known in the art. In the following, thethromboelastograph ROTEG 05 by the company Pentapharm GmbH,Kreillerstraβe 21, D-81673 Munich, Germany, was used.

Description of the thrombelastographic measuring process: The measuringcells of the thrombelastograph by the company Pentapharm GesmbH, Munichconsists of a cuvette and a plug, which are inserted into the measuringdevice. The cuvette is filled with the materials to be tested prior toinsertion, heated to 37° C., and the plug is inserted accordingly.Measurement time is 60-150 min, and the process of clot formation ismonitored using the following parameters:

-   -   a. Coagulation time (CT). CT is the time span from the start        point to the occurrence of an amplitude of 2 mm.    -   b. Clot formation time (CFT). CFT is the time span in which the        amplitude rises from 2 to 20 mm.    -   c. α-angle. α-angle is the parameter which results from the        angle of the mid line and the tangent of the curve of the        amplitude.    -   d. Clot formation rate (CFR). CFR is the widest angle of the        slope of the curve of the amplitude between its tangent and the        midline.    -   e. Maximum clot formation (MCF). MCF is the greatest distance of        the curve of the amplitude from the midline.    -   f. MCF-time (MCF-t). MCF-t is the time span from the time point        of coagulation to the time point of achieving maximum clot        firmness.    -   g. Maximum lysis (ML). ML is defined by the greatest difference        in height between the MCF and the lowest amplitude resulting        from lysis.    -   h. Lysis time (LT). LT is the time span from the time point of        coagulation onset to the time point of clot dissolution.    -   i. Lysis onset (LOT). LOT is the time span from the time point        of coagulation onset to the time point of lysis onset.

240 μl of platelet-rich normal plasma or inhibitor plasma are pipettedinto the cuvette. Other additions of solutions or reagents must notexceed 80 μl, and the volume per cuvette must not exceed 320 μl. If theadditions are less than 80 μl, the cuvette is filled up to 320 μl withisotonic saline.

FIG. 1: Thrombelastogram 3372 was obtained by the addition of a dilutionof relipidated TF of 1:30000 to platelet-rich normal plasma.Thrombelastogram 3373 was obtained by the addition of a dilution ofrelipidated TF of 1:300000 to platelet-rich normal plasma.

FIG. 2: Thrombelastogram 3569 was obtained by the addition of a dilutionof relipidated TF of 1:30000 to factor-VIII inhibitor plasma.Thrombelastogram 3570 was obtained by the addition of a dilution ofrelipidated TF of 1:300000 to factor-VIII inhibitor plasma.

FIG. 3: Thrombelastogram 3611 was obtained by the addition of a dilutionof relipidated TF of 1:30000 to platelet-rich inhibitor plasma, to whichalso 10 U factor VIIa were added. Thrombelastogram 3612 was obtained bythe addition of a dilution of relipidated TF of 1:30000 to platelet-richinhibitor plasma, to which also 1 U factor VIIa was added.Thrombelastogram 3613 was obtained by the addition of a dilution ofrelipidated TF of 1:30000 to platelet-rich inhibitor plasma, to whichalso 1/10th U factor VIIa was added. Thrombelastogram 3614 was obtainedby the addition of a dilution of relipidated TF of 1:30000 toplatelet-rich inhibitor plasma, to which also 1/100th U factor VIIa wasadded.

FIG. 4: Thrombelastogram 3607 was obtained by the addition of a dilutionof relipidated TF of 1:300000 to platelet-rich inhibitor plasma, towhich also 10 U factor VIIa were added. Thrombelastogram 3608 wasobtained by the addition of a dilution of relipidated TF of 1:300000 toplatelet-rich inhibitor plasma, to which also 1 U factor VIIa was added.Thrombelastogram 3609 was obtained by the addition of a dilution ofrelipidated TF of 1:300000 to platelet-rich inhibitor plasma, to whichalso 1/10th U factor VIIa was added. Thrombelastogram 3610 was obtainedby the addition of a dilution of relipidated TF of 1:300000 toplatelet-rich inhibitor plasma, to which also 1/100th U factor VIIa wasadded.

FIG. 5: Thrombelastogram 3677 was obtained by the addition of a dilutionof relipidated TF of 1:30000 to platelet-rich inhibitor plasma, to whichalso a 1:10 dilution of factor XIa concentrate was added.Thrombelastogram 3678 was obtained by the addition of a dilution ofrelipidated TF of 1:30000 to platelet-rich inhibitor plasma, to whichalso a 1:100 dilution of factor XIa concentrate was added.Thrombelastogram 3679 was obtained by the addition of a dilution ofrelipidated TF of 1:30000 to platelet-rich inhibitor plasma, to whichalso a 1:1000 dilution of factor XIa concentrate was added.

FIG. 6: Thrombelastogram 3673 was obtained by the addition of a dilutionof relipidated TF of 1:300000 to platelet-rich inhibitor plasma, towhich also a 1:10 dilution of factor XIa concentrate was added.Thrombelastogram 3674 was obtained by the addition of a dilution ofrelipidated TF of 1:300000 to platelet-rich inhibitor plasma, to whichalso a 1:100 dilution of factor XIa concentrate was added.Thrombelastogram 3675 was obtained by the addition of a dilution ofrelipidated TF of 1:300000 to platelet-rich inhibitor plasma, to whichalso a 1:1000 dilution of factor XIa concentrate was added.

EXAMPLES 1. Thrombin- and Pyrogen-Free Virus Safe Factor XIaConcentrates

10 l sourceplasma will be frozen, thawn and the cryoprecipitate formedafter complete thawing will be removed. The supernatant will be mixedwith 500 g washed DEAE-Sephadex-A50 and stirred for 30 min at 4° C.Sephadex will be removed by filtration and the supernatant may be usedin the production for further plasma fractions. The Sephadex will bewashed 3 times at 4° C. with 10|0,01 molar phosphate buffer, pH 7,8containing 1% NaCl. Factor XI will be eluted with the same phosphatebuffer (2 1) containing 3% NaCl. The eluate will be lightly stirred at30° C. for 12 h with 20 g Tween-80 and 6 g trinitrobutylphospate (TNBP).Tween-80 and TNBP will be removed by—multiple if necessary—adsorptionand elution of Factor XI on DEAE-Sephadex-A50. It will be advantageousto wash the Sephadex adsorbed Factor XI with a 1% buffered NaCl solutionto removal of the solvent and detergents.

The last Sephadex eluate will be diluted 1+4 with 0,01 molar phosphatebuffer, pH 8,2. The Factor XI in this deluted eluate will be adsorbedonto a DEAE-Sephadex-A50 column. After washing the column with 0,1 molarphosphate buffer pH 7,8 containing 1% NaCl, a linear gradient elutionstep will be performed; whereby the NaCl gradient will be prepared bymixing a 0,01 molar phosphate buffer solution, pH 7,5-7,8 with the samephosphate buffer containing 3% NaCl. Fractions of 100 ml each will becollected and the fractions containing Factor XI will be used forfurther processing.

Factor XI can be activated by Factor XIIa, Factor XIa, trypsin, thrombinand other agents. In case of this example with virus safe commerciallyavailable thrombin and heparin the optimal conditions for activatingFactor XI in the concentrates will be determined in preliminaryexperiments at room temperature over 3 h. While the preliminaryexperiments are being performed, the Factor XI concentrate can be keptfrozen at −20 ° C. to avoid bacterial contamination. After the optimalamounts of thrombin and heparin for activation have been determined, thefrozen Factor XI concentrate will be thawn and activated with thedetermined amounts of thrombin and heparin at 10° C. for 4 h.

The Factor XIa containing solution will be depleted of thrombin byrepeating adsorption and elution of Factor XIa on DEAE-Sephadex-A50, insuch a way that the last DEAE-Sephadex-eluate should not contain morethan 0,1 units of thrombin per ml (i.e. 0,01 units of thrombin per 100pl), which is regarded as “thromin-free” in the sense of the presentdescription and claims. The concentration steps of Factor XIaconcentrates including the removal of solvent detergents as well as theconcentration of Factor XIa activating agents can also be performed by agradient elution of Factor XIa with other resins adsorbing Factor XIasuch as QAE-Sepharose, SP-Sepharose or Concanavalin-A-Sepharose.

For viral safety the factor XIa concentrate will be further processed bynano filtration applying a 20 nm pore filter. Before this filtration itmay be necessary to clarify the solution by a 75 nm and a 35 nm porefilter as well as by a protein permeable ultrafilter. It might beadvantages to ultracentrifuge the solution in a flow through centrifugewith a minimun of 50.000 rpm. After adequate formulation the Factor XIawill be filtered through a sterile filter of 100 or 200 nm pore size andthe sterile bulk solution will be filled in final containers and freezedried. The humidity content of the freeze dried Factor XIa concentrateshould be 0,8-2,0% water. The freeze dried Factor XIa in the finalcontainers will undergo heat treatment after air-tight sealing for 30min at 100° C. as an additional virus inactivation step. This is apreferred embodiment of the invention.

2. Proof of Absence of Thrombin in the Factor XIa Concentrate:

100 μl of factor a XIa concentrate will be mixed with 200 μl of 0,1%fibrinogen solution; both solutions will be adjusted at a pH 7,8 and atemperature of 37° C. The exact time of mixing will be recorded and themixture will be observed for 250 sec to register clotting and/orflocculation. In parallel 100 μl of 0,1 unit thrombin per ml solutionwill be mixed with 200 μl of 0,1% fibrinogen solution at 37° C. pH 7,5and under keeping the temperature at 37° C. will be observed for 250sec. Within this time period complete clotting of the fibrinogen addedis expected to occur. The factor XIa concentrate is determined as“thrombin free” if after mixing with fibrinogen neither clotting norflocculation occurs and if in parallel the 0,01 unit thrombin withfibrinogen clots.

3. Estimation of the Factor XIa Activity:

Activity will be assessed with the chromogenic substrate S2366 at 37° C.in a buffered solution of pH 8,3. Dilutions of a Factor XI concentrateor a Factor XIa concentrate will be determined in a volume of 200 μl. 15μl of 30 mmol S2366 solution will be added and adjusted with TRIS bufferpH 8,3 to a total volume of 300 μl and mixed. The tests will beperformed in micro titer plates and each well covered with 2 drops ofmineral oil. Extinction will be followed for 2 h and the activity ofFactor XIa in the concentrate will be estimated based on thedose-response-curve of a Factor XIa reference preparation.

4. Platelet-Rich Normal or Factor-VIII Inhibitor Plasma:

Platelet-rich plasma from healthy donors was centrifuged at 4000revolutions per minute for 5 minutes, the supernatant plasma wasremoved, and the platelets were washed with a phosphate glucose bufferof pH 6.5 three times. The last sediment was suspended in platelet-poornormal plasma or platelet-poor factor-VIII inhibitor plasma, and theplatelet suspension was adjusted to a platelet count between two andthree million platelets per μl. This platelet concentrate was dilutedprior to use 1:10 with platelet-poor normal plasma or platelet-poorfactor-VIII inhibitor plasma. The platelet-poor factor-VIII inhibitorplasma must have a content of at least 100 Bethesda Units per ml.

In the sample for thrombelastogram 3372 (FIG. 1), 240 μl ofplatelet-rich normal plasma were mixed with 10 μl of a TF dilution of1:30000. After the addition of 50 μl of isotonic saline, coagulation wasinitiated by the addition of 20 μl of a 0.2 M CaCl2 solution.

The same procedure was used with sample 3373 (FIG. 1), except that theTF solution used was diluted 1:300000.

As can be seen from the thrombelastogram (FIG. 1), CT was 673 sec, andafter the addition of a an amount of TF to 1/10^(th), was 659 sec,resulting in a non-significant difference. MCF was 63 ml with bothsamples. Equally, the difference in the α-angle was not significant.

When platelet-rich factor-VIII inhibitor plasma was used, there weresignificant differences with different TF concentrations. The CT insamples 3569 and 3570 (FIG. 2) was 1523 sec. in both when a TF dilutionof 1:30000 was used and was much delayed compared to platelet-richnormal plasma, and was still considerably prolonged in sample 3570,which gave 2588 sec.

Also MCF in samples 3569 and 3570, which was 38 mm and 27 mm,respectively, was much reduced compared to samples 3372 and 3373. Theseresults suggest that the TF dilution of 1:300000 gives the greatestdifference between the coagulation behavior of normal plasma and that ofplatelet-rich inhibitor plasma.

The glucose-phosphate buffer used had the following composition:

sodium citrate dihydrate 2.2%

citric acid 0.8%

glycose monohydrate 2.42%

RecombiPlas Tin-Hemosil lot NO574097G from Instr.Lab. was used. Thenormal plasma was freshly obtained donor plasma. The factor-VIIIinhibitor plasma was lot 824 and was supplied by Biomex.

5. Normalization of the Thrombelastographic Coagulation Parameters ofPlatelet-Rich Factor-VIII Inhibitor Plasma by the Addition ofFactor-VIIa Concentrate using TF Dilutions of 1:30000 and 1:300000:

As can be seen from FIG. 3, the coagulation parameters of platelet-richfactor-VIII inhibitor plasma were only normalized by the addition of 10U of factor VIIa (thrombolastogram 3611). The addition of 1 U (3612),1/10^(th) (3613) and 1/100^(th) (3614) of a U (prolonged the coagulationtime to 1153 sec, 2161 sec, and 2542, respectively. 10 U of factor VIIanormalized the MCF completely to 64 mm, while all additions with lowerVIIa gave a gradual MCF shortening.

When a higher TF dilution of 1:300000 was used (FIG. 4), practically allcoagulation parameters could be normalized with the addition of 10 U(3607) of factor VIIa. However, the difference in the CT was morepronounced than with a lower TF dilution, the addition of 1 U (3608), of1/10^(th) (3609) and 1/100^(th) (3610) of a U prolonging CT to 1265 sec,2581 sec, and 4749 sec, respectively.

The factor VII concentrate used was lot SU61347 by Novo Nordisk.

6. Normalization of the Thrombelastographic Coagulation Parameters ofPlatelet-Rich Factor-VIII Inhibitor Plasma by the Addition of aFactor-XIa Concentrate using TF Dilutions of 1:30000 and 1:300000:

The same samples and reagents were used as in Example 2, except that anS/D virus inactivated, nanofiltered factor XIa concentrate was usedinstead of a factor VIIa concentrate, which was heated in freeze-driedstate at 100° C. for 30 min and which yielded a solution of factor XIaof 1100 U per ml after reconstitution with WFI.

As can be seen from thrombelastogram 3677 (FIG. 5), the CT of 379 sec.was significantly shorter with 1/10^(th) of a U of factor XIa than thatof normal plasma (FIG. 1: 3372). With 59 mm, the MCF was also in thenormal range. The same was true for an addition of only 1/100^(th) of aU of factor XIa, which gave a CT in the normal range. See Sample 3678(FIG. 5). As can be seen from sample 3679, only 1/1000^(th) of a U offactor XIa prolongs the CT to 1625 sec.

When the higher dilution of TF of 1:300000 was used along with factorXIa, the normalization of the coagulation parameters was even morepronounced than with factor VIIa concentrates.

REFERENCES CITED

Astermark J, et al. A randomized comparison of bypassing agents inhemophilia complicated by an inhibitor: the FEIBA NovoSeven Comparative(FENOC) Study. Blood, 2007;109:546-551.

Brown S, Haemostasis from bench to bedside. Haemophilia. 2002; 8:1-9Eibl J et al., AT350726, 1976.

Fekete L, et al. ‘Auto’ factor IX concentrate: a new therapeuticapproach to treatment of hemophilia A patients with inhibitors.International Congress of Hematology, Sao Paulo, 1972;295 [Abstract].

Hedner U, EP0225160B1,1986.

Hedner U, et al. Use of human factor VIIa in the treatment of twohemophilia A patients with high-titer inhibitors. J Clin Invest.1983;71:1836-41.

Kelly P, et al. Antihemophilic factor inhibitors. Management withprothrombin complex concentrates. JAMA, 1976;236:2061-4.

Kumar R, et al. The Influence of Fibrinogen and Fibrin on ThrombinGeneration—Evidence for Feedback Activation of the Clotting System byClot Bound Thrombin. Thrombosis and Haemostasis 1994;72:713-21.

Kurczynski E, et al. Activated prothrombin concentrate for patients withfactor VIII inhibitors. N Engl J Med, 1974;291:164-7.

Mann K G, et al. Blood Coagulation Dynamics in Haemostasis.Hamostaseologie 2009;29:7-16.

Roberts H, et al. The use of recombinant factor VIIa in the treatment ofbleeding disorders. Blood.2004;104:3858-3864.

Sixma J, et al. The haemostatic plug in haemophilia A: a morphologicalstudy of haemostatic plug formation in bleeding time skin wounds ofpatients with severe haemophilia A. British Journal of Haematology,1984; 58:741-753.

Turecek P, et al. FEIBA: mode of action. Haemophilia.2004;10:(Suppl.2):3-9 van den Berg, H, et al. Clinical Prediction Modelsfor Inhibitor Development in Severe Hemophilia A. J. Thrombosis andHaemostasis, 2009;7 (Suppl.1):98-102.

Walsh P. Roles of Platelets and Factor XI in the Initiation of BloodCoagulation by Thrombin. Thromb Haemost 2001;86:75-82.

1. A thrombin-free factor-XIa concentrate or a thrombin-free coagulationfactor concentrate containing factor XIa for the manufacture of apharmaceutical preparation which does not form thrombin during storage,and for the manufacture of a parenterally applicable drug product whichdoes not form thrombin during or after freeze-drying, storage andreconstitution.
 2. The pharmaceutical preparation according to claim 1,which contains serpins and/or high molecular weight kininogen or itssplit products.
 3. The pharmaceutical preparation according to claim 1,which is nanofiltered to improve viral safety, either before or afterformulation.
 4. The pharmaceutical preparation according to claim 1,which is rendered virally safe by treatment with solvent/detergent andsubsequent removal of solvent and detergent.
 5. The pharmaceuticalpreparation according to claim 1, which is filled under sterile andpyrogen-free conditions and is freeze-dried and virally inactivated byheating the filled, freeze-dried pharmaceutical preparations at no lessthan 90° C. and no more than 140° C. for 30 to 180 minutes. 6.Thrombin-free factor-XIa concentrate or thrombin-free coagulation factorconcentrate containing factor XIa according to claim 1, which isobtained by fractionation of plasma or serum or by genetic engineering.7. Thrombin-free factor-XIa concentrate or thrombin-free coagulationfactor concentrate containing factor XIa according to claim 1, which arevirally safe and pyrogen-free.
 8. A diagnostic kit comprising: one ormore components configured for the determination of the amount offactor-XIa concentrate which is necessary to normalize prolonged bloodcoagulation in a reference factor-VIII inhibitor plasma and in bloodsamples of patients with coagulation disorders.
 9. A method ofmanufacturing a medicinal drug product for treatment of coagulationdisorders, the method comprising: providing the thrombin-free factor-XIaconcentrate or a thrombin-free coagulation factor concentrate containingfactor XIa of claim 1; and preparing a medicinal drug product from thethrombin-free factor-XIa concentrate or a thrombin-free coagulationfactor concentrate containing factor XIa as an active ingredient. 10.The pharmaceutical preparation of claim 3, wherein the nanofilteringuses a nanofilter with a pore diameter of ≦20 nm.
 11. The pharmaceuticalpreparation of claim 3, wherein the nanofiltering uses a nanofilter is a1000 kDalton ultrafilter.
 12. A pharmaceutical preparation comprising: athrombin-free factor-XIa concentrate or a thrombin-free coagulationfactor concentrate containing factor XIa which does not form thrombinduring storage.
 13. The pharmaceutical preparation of claim 12, whereinthe pharmaceutical preparation is configured as a parenterallyapplicable drug product which does not form thrombin during or afterfreeze-drying, storage and reconstitution.
 14. The pharmaceuticalpreparation according to claim 12, which contains serpins and/or highmolecular weight kininogen or its split products.
 15. The pharmaceuticalpreparation according to claim 12, which is nanofiltered to improveviral safety, either before or after formulation.
 16. The pharmaceuticalpreparation of claim 15, wherein the nanofiltering uses a nanofilterwith a pore diameter of ≦20 nm.
 17. The pharmaceutical preparation ofclaim 15, wherein the nanofiltering uses a nanofilter is a 1000 kDaltonultrafilter.
 18. The pharmaceutical preparation according to claim 1,which is rendered virally safe by treatment with solvent/detergent andsubsequent removal of solvent and detergent.
 19. The pharmaceuticalpreparation according to claim 1, which is filled under sterile andpyrogen-free conditions and is freeze-dried and virally inactivated byheating the filled, freeze-dried pharmaceutical preparations at no lessthan 90° C. and no more than 140° C. for 30 to 180 minutes.
 20. Thepharmaceutical preparation according to claim 1, which is virally safeand pyrogen-free.